I. M-Protein PA0 II. Electrophoretic Immunosubtraction
Antibodies are a class of immunoglobulins that are capable of recognizing and binding "foreign" (or antigenic) molecules that invade mammals and other vertebrates. A typical antibody consists of a pair of two "heavy" chains linked to a pair of two identical "light" chains to form a hypothetical "Y" structure. The heavy chains form the base of the "Y," and the light chains form the two branches. The heavy chains and light chains are separately synthesized by the immune system. There are two types of light chains, referred to as "kappa" (".kappa.") and "lambda" (".lambda.") . Similarly, there are several classes of heavy chains: .gamma. ("IgG"); .alpha. (IgA); .delta. ("IgD"); .mu. ("IgM") and .epsilon. ("IgE"). IgG, IgA and IgM are the major serum immunoglobulins; IgD and IgE are generally present in serum only at very low concentrations.
The IgM immunoglobulin differs from other classes of immunoglobulins in that it is a pentamer of the basic four-chain antibody. It thus contains five "Y" structures, each of which has 2 light and 2 heavy chains. The monomeric units of IgM are held together by disulfide bonds, and by a single polypeptide, known as the "J chain." Free, monomeric M-protein is not found in healthy individuals, but rather is a distinguishing feature of a number of diseases and conditions (Bush, S. T. et al., J. Lab. Clin. Med. 73:194-201 (1969)).
Indeed, in healthy individuals, the synthesis of the antibody chains is synchronized by the immune system, such that under normal circumstances, only complete immunoglobulins are produced. Upon encountering an antigen, the B-cells of the immune system clonally proliferate to ensure the production of a sufficient amount of immunoglobulins to neutralize the invading antigen. After the invasion has been neutralized, the production of the immunoglobulins typically ceases. Occasionally, however, unregulated B-cell clones will escape regulation, and continue to produce immunoglobulin even after the antigen has been eliminated. The immunoglobulins of such cells have the same antigen binding site, suggesting that the cells reflect the clonal proliferation of a single ancestor cell. The immunoglobulins produced by such cells are referred to as monoclonal immunoglobulins, or "M-proteins."
The production of such M-proteins can reflect the presence of serious disease. Multiple myeloma, for example, is associated with the production of IgG, IgA, IgD, IgM or IgE M-proteins. A major pathologic feature of multiple myeloma is bone destruction, i.e., bone deformity or acute, painful pathological fractures. Clinically, the patient may experience bone pain, infections due to decreased production of normal Ig's, and anemia. Twenty percent of myeloma patients evidence Bence Jones protein, which is a free monoclonal light chain. Multiple myeloma can also impact neural tissue (i.e., the spinal cord, nerve roots and cranial or peripheral nerves).
The production of IgM M-proteins is associated with rheumatoid arthritis, certain immunodeficiency diseases, infective diseases, and B cell lymphoproliferative disorders, such as multiple myeloma, Waldenstr om's macroglobulinemia and lymphoma (Ishii, H., Acta. Med. Okayama 42:279-286 (1988); Roberts-Thomson, P. J. et al., Austr. NZ J. Med. 14:121-125 (1984); Carter, P. M. et al., Br. Med. J. 2:260-261 (1971); Harris-Dangkul, V. et al., J. Immunol. 155:216-222 (1977)). Its presence in such diseases may amount to between 10-40% of the total IgM concentration (Roberts-Thomson, P. J. et al., Austr. NZ J. Med. 14:121-125 (1984)), and the overall expression of the protein appears to correlate with the clinical significance of such diseases (Nagai, K. et al., Scand. J. Immunol. 14:99-108 (1981); Ishii, H., Acta. Med. Okayama 42:279-286 (1988)). The increased production of IgM increases the viscosity of the patient's blood (causing "hyperviscosity"), and is associated with headache, dizziness and vertigo.
The heavy chain composition (i.e., IgA, IgG, IgM, IgE, IgD) of an M-protein defines that protein's class. The light chain composition (.kappa. or .lambda.) of the protein defines its type. The classification and typing of an M-protein is of substantial clinical value and importance, and a variety of approaches have been used to classify and type M-proteins. For example, serum M-protein levels have been determined using column chromatography (Sugai, S. et al., Jpn. J. Clin. Oncol. 13:533-542 (1983); Roberts-Thomson, P. J. et al., Austr. NZ J. Med. 14:121-125 (1984)), cellulose acetate electrophoresis (Ishii, H., Acta. Med. Okayama 42:279-286 (1988); Sezaki, T. et. al., Jpn. J. Clin. Hematol. 23:847-853 (1982)), agarose gel electrophoresis (Beckman Instruments Inc. Paragon System), capillary electrophoresis methods (U.S. Pat. No. 5,228,960), and by hemolytic plaque assays (Nagai, K. et al., Scand. J. Immunol. 14:99-108 (1981)). An ELISA for M-proteins has also been developed (Sugai, S. et al., Jpn. J. Clin. Oncol. 13:533-542 (1983)). In this assay, M-protein is coupled to glass beads and permitted to react with rabbit anti-M-protein antibodies, followed by reaction with peroxidase-conjugated goat anti-rabbit IgG.
The analysis of M-proteins has been facilitated by electrophoretic methods. Such methods exploit the fact that proteins in solution have an intrinsic electrical charge. In the presence of an electric field, this intrinsic charge imparts a characteristic "electrophoretic" mobility to the protein, and thus permits various species of proteins to separate from one another. Under the influence of such a field, all of the proteins will move toward a designated electrode having a charge opposite to the charge of the proteins; those proteins having a lower electrophoretic mobility will move slower than, and hence be separated from, those proteins having a (relative) higher electrophoretic mobility.
Immunological electrophoretic methods, such as Immunofixation electrophoresis ("IFE"), Immunoelectrophoresis ("IEP"), and especially Immunosubtraction Electrophoresis ("ISE") have been used to classify and type M-proteins.
IEP and IFE are related procedures (Beckman Bulletin EP-2. "Immunoelectrophoresis Applications Guide." (1991)). IFE is a two stage procedure using agarose gel protein electrophoresis in the first stage and immunoprecipitation in the second. In a clinical setting for the analysis of immunoglobulins, a clinical sample (e.g., whole blood, serum, plasma, urine, cerebrospinal fluid) is placed in multiple positions ("lanes") on an agarose gel. Because immunoglobulins are proteins, they have a charge distribution such that when an electric field is applied to the gel-containing sample, the immunoglobulins will traverse the gel from anode to cathode. Thereafter, antisera comprising antibodies to specific immunoglobulin classes and types (typically IgG, IgA, IgM, kappa and lambda) are applied to specific lanes. The gel and antisera are incubated, during which time immune complexes form between the specific immunoglobulins and the antibodies. The location of such immune complexes are visualized by staining. By using a reference pattern on the gel, one can then determine the type of immunoglobulin present on the gel. The presence of a particular band is thus indicative of the presence of an M-protein corresponding to a particular immunoglobulin type. Methods of conducting IFE are disclosed by Chen, F-.T. A., U.S. Pat. No. 5,202,006; Chen, F-.T. A., U.S. Pat. No. 5,120,413; all herein incorporated by reference).
The PARAGON.RTM. electrophoresis system (Beckman Instruments, Inc., Fullerton, Calif., U.S.A.) is a commercially available system for conducting both IFE and IEP (See also, Gebott, et al., U.S. Pat. No. 4,669,363; Beckman Bulletin EP-3 "Paragon.RTM.Serum Protein Electrophoresis II (SPE-II) Applications Guide" (1990); Beckman Bulletin EP-2. "Immunoelectrophoresis Applications Guide" (1991); Beckman Bulletin EP-4 "Immunofixation Electrophoresis Applications Guide" (1991); Beckman Instructions 015-246513-H "Paragon.RTM. Electrophoresis System-IFE" (1990); Beckman Bulletin EP-6 "High Resolution Electrophoresis in the Detection of Monoclonal Gammopathies and Other Serum Protein Disorders." (1990); Chen, F-.T. A. et al. Clin. Chem. 37:14-19 (1991)).
Like immunoelectrophoresis, immunosubtraction electrophoresis (ISE) is a variation of IFE (Aguzzi, F. et al., Estratto dal. Boll. 1st Sieroter, Milanese 56:212-216 (1977); White, W. A. et al., Biochem. Clin. 10:571-574 (1986); Merlini, G. et al., J. Clin. Chem. Biochem. 21:841-844 (1983); Liu, C-.M. et al., U.S. Pat. No. 5,228,960, herein incorporated by reference). In ISE, however, the sample is pretreated with an insolubilized antibody directed to a particular "target" protein. If the target protein is present, it will bind to the antibody and thus be removed from the sample. The sample is then applied to a gel and subjected to electrophoresis. If the target protein had been present in the initial sample, visualization of the proteins in the gel would reveal a "negative band (i.e. an absence of staining) at the position in the gel where the removed band would have migrated to, had it not been removed by the antibody. Thus, the absence of a particular band is indicative of the presence of the corresponding target protein in the sample.
IEP, IFE and ISE thus each require multiple steps, and the preparation and use of a separation gel and a signal-generating stain. The labor intensive nature of these procedure is an obvious impediment in a clinical setting. Additionally, the amount of disposable end-products associated with these procedures can further increase the allied costs associated with these procedures.
In view of the deficiencies of these methods in clinical settings, less labor-intensive methods that permit greater throughput with lower cost have been sought. One such method is "Capillary Zone Electrophoresis" ("CZE") (Chen, F-.T. A., et al., Clin. Chem. 77:14-19 (1991); U.S. Pat. No. 5,120,413, both herein incorporated by reference). Capillary zone electrophoresis permits rapid and efficient separations of proteins (Grossman, P., et al., Anal. Chem. 61:1186-1194 (1989)), and other charged substances. Separation of the constituents of clinical samples can typically be accomplished in less than 20 minutes, typically in less than 10 minutes.
In general, CZE involves introducing a sample into a capillary tube, i.e. a tube having an internal diameter of from about 2 .mu.m to about 200 .mu.m (preferably, less than about 50 .mu.m, most preferably, about 25.mu.m or less), and applying an electric field to the tube. The electric potential of the field pulls the sample through the tube and separates it into its constituent parts. Since each of the sample constituents has its own individual electrophoretic mobility, those having greater mobility travel through the capillary tube faster than those with slower mobility. Hence, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones.
CZE can be generally separated into two categories based upon the contents of the capillary columns. In "gel" CZE, the capillary tube is filled with a suitable gel, e.g., polyacrylamide gel, and separation of the constituents of the sample is thus predicated by both the size and the charge of the constituents. Despite the speed of analysis, gel CZE has several disadvantages, notably, the unpredictability and non-durable nature of the gel material. These factors make gel CZE unacceptable in any setting where numerous analytical runs are conducted.
In the second form of CZE (i.e. "open" CZE), the capillary tube is filled with an electrically conductive buffer solution (Kim, J. W. et al., Clin. Chem. 39:689-692 (1993)). The capillary tube is then ionized with a negative charge. Such ionization causes the capillary wall to become negatively charged, thereby attracts positive ions from the buffer. Because the electroneutrality of the solution must be maintained, any flow of positive ions towards the capillary wall will be accompanied by a similar movement of the buffer solution and the constituents of the sample. This electroendosmatic flow provides a fixed velocity component which drives both neutral species and ionic species, regardless of charge, towards the cathode. The buffer in "open CZE" is stable against conduction and diffusion. Accordingly, separations can be obtained in "open CZE" that are quite similar to those obtained in gel-based electrophoresis.
Fused silica is principally utilized as the material for the capillary tube because it can withstand the relatively high voltage used in CZE, and because the inner walls ionize to create the negative charge which causes the desired electroosmotic flow. However, alumina, beryllium, Teflon.RTM.-coated materials, glass, quartz and combinations of these (with or without fused silica) can also be utilized. The capillary column is typically capable of withstanding a wide range of applied electrophoretic fields of between about 10 v/cm to about 1000 v/cm. The capillary column may be coated on the outside (using, e.g., a polyimide material) for ease of handling. The inner wall of the capillary may be untreated or coated with a material capable of reducing adsorption to the inner wall during electroosmotic flow of the bulk solution. However, it is typically preferred that the inner wall be uncoated because typical coatings have a tendency to breakdown in an unpredictable manner. In U.S. Pat. No. 5,120,413, analysis of clinical samples was conducted using untreated capillary columns.
Open CZE has many desirable qualities ford e.g., clinical sample analysis: because the analysis does not involve a gel-filled column, the inherent limitations on the number of analytical runs that can be conducted with any particular gel-filled column are avoided; when the capillary column is untreated, the aura of unpredictability which can be associated with coated columns is avoided; the sample size is small (usually on the order of 5 to 200 nL of diluted sample); sample analysis time is fast, i.e. less than about 20 minutes; and the protocol lends itself to automation, thus decreasing the labor skills necessary for efficient and effective sample analysis.
Liu, C-.M. et al., U.S. Pat. No. 5,228,960, which has been incorporated by reference herein describes a recent modification to the CEI procedure that facilitates the classifying and typing of M-proteins. In the method, a portion of the sample is incubated with an insolubilized or insolubilizable binding partner that is capable of substantially removing M-protein from the solution, and then subjected to CZE. The "electropherogram" (i.e. a graphical representation of the separation of the constituent parts of the sample) of that portion is compared with that of an untreated sample. If the sample contains an M-protein, the electropherogram of the untreated sample will evidence the "complete" constituent profile of the sample; similarly, the electropherogram of the treated sample, when compared to the first, will evidence a "subtracted" peak corresponding to the M-protein that was removed from the sample.
As is evident, the method requires the use of a solid phase to effect the separation of the M-protein from the sample. The use of this heterogeneous system increases the incubation time of the sample, and therefore lowers the throughput of the analysis. It has not been possible to omit such a separation step because antibodies, and in particular, IgGs, the major serum immunoglobulins, co-migrate with heavy chain M-proteins. The amount of IgG in normal serum is larger than 20 mg/mL and the level of IgA and IgM is greater than 10 mg/mL. (see, for example, Roberts-Thomson, P. J. et al., Austr. NZ J. Med. 14:121-125 (1984)).
In view of the importance of accurately classifying and typing M-proteins, it would be desirable to possess a technique applicable to such analysis that would provide results with a minimum of processing steps, be easy to use, have high throughput and would avoid the end-product disposal problem occasioned by the use of separating gels.